Compositions of active WNT protein

ABSTRACT

Compositions of purified biologically active Wnt proteins are provided. Wnt proteins are found to be hydrophobic and post-translationally modified by addition of a lipid moiety at a conserved cysteine residue. Methods for isolation of Wnt utilize detergents that maintain the solubility of the modified protein.

CROSS REFERENCE

This application is a Continuation of U.S. application Ser. No.14/966,930, filed Dec. 11, 2015, which is a Division of U.S. applicationSer. No. 14/824,930, filed Aug. 12, 2015, now U.S. Pat. No. 9,441,198,which is a Division of U.S. application Ser. No. 14/157,331, filed Jan.16, 2014, now U.S. Pat. No. 9,139,636, which is a continuation of U.S.application Ser. No. 12/480,550, filed Jun. 8, 2009, now U.S. Pat. No.8,642,335, which is a Division of U.S. application Ser. No. 11/796,186,filed Apr. 27, 2007, now abandoned, which is a Division of U.S.application Ser. No. 10/816,720, filed Apr. 1, 2004, now U.S. Pat. No.7,153,832, which claims priority to U.S. Provisional Patent ApplicationSer. No. 60/461,167, filed Apr. 7, 2003, which applications areincorporated herein by reference in their entirety.

INTRODUCTION

Wnt proteins form a family of ihighly conserved secreted signalingmolecules that regulate cell-to-cell interactions during embryogenesis.Wnt genes and Wnt signaling are also implicated in cancer. Insights intothe mechanisms of Wnt action have emerged from several systems: geneticsin Drosophila and Caenorhabditis elegans; biochemistry in cell cultureand ectopic gene expression in Xenopus embryos. Many Wnt genes in themouse have been mutated, leading to very specific developmental defects.As currently understood, Wnt proteins bind to receptors of the Frizzledfamily on the cell surface. Through several cytoplasmic relaycomponents, the signal is transduced to beta-catenin, which then entersthe nucleus and forms a complex with TCF to activate transcription ofWnt target genes.

Wnt glycoproteins are thought to function as paracrine or autocrinesignals active in several primitive cell types. The Wnt growth factorfamily includes more than 19 genes identified in the mouse and inhumans. The Wnt-1 proto-oncogene (int-1) was originally identified frommammary tumors induced by mouse mammary tumor virus (MMTV) due to aninsertion of viral DNA sequence (Nusse and Varmus (1982) Cell31:99-109). Expression of Wnt proteins varies, but is often associatedwith developmental process, for example in embryonic and fetal tissues.Wnts may play a role in local cell signaling. Biochemical studies haveshown that much of the secreted Wnt protein can be found associated withthe cell surface or extracellular matrix rather than freely diffusiblein the medium.

Studies of mutations in Wnt genes have indicated a role for Wnts ingrowth control and tissue patterning. In Drosophila, wingless (wg)encodes a Wnt gene and wg mutations alter the pattern of embryonicectoderm, neurogenesis, and imaginal disc outgrowth. In Caenorhabditiselegans, lin-44 encodes a Wnt, which is required for asymmetric celldivisions. Knock-out mutations in mice have shown Wnts to be essentialfor brain development, and the outgrowth of embryonic primordia forkidney, tail bud, and limb bud. Overexpression of Wnts in the mammarygland can result in mammary hyperplasia, and precocious alveolardevelopment.

Wnt signaling is involved in numerous events in animal development,including the proliferation of stem cells and the specification of theneural crest. Wnt proteins are therefore potentially important reagentsin expanding specific cell types, but in contrast to other developmentalsignaling molecules such as the Hedgehogs and the BMPs, Wnt proteinshave never been isolated in an active form. Although Wnt proteins aresecreted from cells, secretion is usually inefficient and previousattempts to characterize Wnt proteins have been hampered by their highdegree of insolubility.

Publications

The biological activity of soluble wingless protein is described in vanLeeuwen et al. (1994) Nature 24:368(6469):342-4. Biochemicalcharacterization of Wnt-frizzled interactions using a soluble,biologically active vertebrate Wnt protein is described by Hsieh et al.(1999) Proc Natl Acad Sci USA 96(7):3546-51. Bradley et al. (1995) MolCell Biol 15(8):4616-22 describe a soluble form of wnt protein withmitogenic activity.

SUMMARY OF THE INVENTION

Compositions of purified and biologically active Wnt proteins areprovided, as well as methods for producing such compositions. Wntproteins are found to be hydrophobic and post-translationally modifiedby addition of a lipid moiety at a conserved cysteine residue. Thislipid modification is important for the biological activity of the Wntprotein, although the unmodified form has some activity at highconcentrations.

Purified and biologically active Wnt compositions are prepared byexpression of a Wnt protein, preferably a secreted form of a Wntprotein. In one embodiment of the invention, the Wnt protein is producedin a palmitoylated form. The presence of the palmitate causes Wnt to berelatively insoluble, and so isolation steps are preferably performed inbuffer containing a concentration of detergent sufficient to maintainsolubility. A first step in purification is dye ligand chromatography.The purified protein fraction can then be further purified by sizeexclusion chromatography, and by cation exchange chromatography. Themethods provide for a substantially homogeneous composition ofbiologically active Wnt protein.

The purified Wnt compositions find use in a variety of therapeuticmethods, including the maintenance and growth of stem cells, tissueregeneration, and the like. In another embodiment of the invention,methods are provided for inhibiting Wnt activity by interfering with thelipid modification of Wnt.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. The patent orapplication file contains at least one drawing executed in color. Copiesof this patent or patent application publication with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee. It is emphasized that, according to common practice, the variousfeatures of the drawings are not to-scale. On the contrary, thedimensions of the various features are arbitrarily expanded or reducedfor clarity. Included in the drawings are the following figures.

FIG. 1A-1D. Wnt3A and DWnt8 Purification. FIG. 1A Coomassie staining ofan SDS polyacrylamide gel containing fractions from all steps of thepurification reveals the enrichment of the Wnt3A protein. Also shown isthe final DWnt8 fraction, purified using the same protocol. Size markersare in kilodaltons. FIG. 1B Wnt3A stabilizes the β-catenin protein.Wnt3A conditioned medium (200 ng/ml) and purified Wnt3A (100 μg/ml) wasdiluted as indicated in medium containing 10% FBS and detected byWestern blot. L cells were stimulated for 2 hours. FIG. 1C Wnt3A inducesexpression of Siamois and Xnr3 in animal cap explants of Xenopusembryos. Animal cap explants were incubated with 100 ng/ml Wnt3A andanalyzed by RT/PCR for expression of the direct targets Xnr3 andSiamois. FIG. 1D Wnt3A induces the morphological transformation of C57MGcells. C57MG cells were treated or not treated with 100 ng/ml Wnt3A for2 days in serum containing medium and then an additional 2 days in serumfree medium.

FIG. 2A-2E. Wnt proteins are palmitoylated on an essential cysteine.FIG. 2A Triton X-114 phase separation (Western blot). A majority of thewild-type Wnt3A separates to the Triton X-114 phase, indicating that itis hydrophobic, but the Wnt3A (C77>A) mutant (see D) partitions mostlyto the water-phase. BSA from serum partitions, as expected, to thewater-phase and serves as an internal control. FIG. 2B In vivo labelingof Wnt3A protein with tritiated palmitate. Wnt3A was partially purifiedfrom conditioned medium of cells labeled with tritiated palmitate for 5hours. FIG. 2C Acyl-Protein Thioesterase (APT-1) treatment of Wnt3A(Western blot). Treatment of Wnt3A with increasing amounts of APT-1shifts the Wnt3A protein from the Triton X-114 phase to the water phase(middle panel) and abolishes its activity in the β-catenin stabilizationassay. FIG. 2D Mass Spectrometry maps the palmitate modification to acysteine (bold) in Wnt3A (C77) and in DWnt8 (C51). The underlinedsequence corresponds to the peptide identified in the spectra as beingmodified. The cysteine is conserved in all Wnt proteins known. Asite-directed mutant (Wnt3A (C77>A) was made and used in FIG. 2A andFIG. 2E. The Drosophila wg S21 allele has a mutation converting thecysteine into a tyrosine and the egl-20 N585 allele in C. elegans has aserine instead of the cysteine. These are both loss of function alleles.FIG. 2E The Wnt3A (C77>A) mutant protein is secreted from 293 cells atlevels similar to wild type, but is not active in increasing β-cateninin target L cells over a range of concentrations tested (Western blot).However, the 293 cells transfected with the Wnt3A (C77>A) expressionconstruct show a modest increase in β-catenin, indicating that highlevels of the mutant can activate. The Wnt3A (C77>A) and wild typetransfected cells express equal levels of Wnt protein.

FIG. 3A-3F. HSCs maintain self-renewing fate with reduceddifferentiation in response to purified Wnt3A. Purified mouse bonemarrow HSCs (c-kit+, Sca-1+, Thy1.1_(lo), Lin−) from Bcl-2 transgenicmice were sorted by FACS and plated as single cells into 60 wellTerasaki plates. Cells were incubated in X-vivo 15 (Bio Whittaker)containing either purified Wnt3A (at approx. 100 ng/ml) plus limitingamounts of SLF (7.5 ng/ml) or SLF (7.5 ng/ml) alone, as a control. FIG.3A Cell growth was monitored over a period of seven days in culture, andshown as the frequency of responding cells. FIG. 3B The total cellgrowth. These cells responded to Wnt3A by proliferating more than a 100fold (from 1 cell to at least 100 cells) and the total number of cellsgenerated was 6 fold greater in the presence of Wnt3A compared tocontrol conditions. Results shown are representative of four independentexperiments. FIG. 3C To determine phenotypic characteristics, cells wereplated in 96 well plates and incubated in the presence of purified orunpurified Wnt3A. After seven days in culture, a majority of cellstreated with purified Wnt3A (at 100 ng/ml) were negative for lineagemarkers (solid line) while a majority treated with unpurified Wnt3A(calculated to be at 200 ng/ml in the medium; table 1) stronglyupregulated Lineage markers (dashed line). FIG. 3D FACS analysis of thepurified Wnt3A treated cells demonstrated that the lineage negativepopulation was distributed into c-Kit⁺ and Sca-1⁺ HSC and c-kit⁺ andSca-1⁻ myeloid progenitors. FIG. 3E Purified mouse bone marrow HSCs(c-kit⁺, Sca-1⁺, Thy1.1^(lo), Lin⁻) were plated singly into 60 wellTerasaki plates and treated with Wnt3A for 6 days following which allcells generated from the single cell were transplanted individually intolethally irradiated recipient mice along with 300,000 rescuing bonemarrow cells. FIG. 3F Peripheral blood (PB) from each transplanted mousewas analyzed after 6 weeks for reconstitution along both lymphoid (B andT) and myeloid (M) lineages. Based on the reconstitution efficiency ofsingle transplanted HSCs it has been estimated that 1/10 (10%) restingHSCs and probably 1/50 (2%) cycling HSCs reconstitute. Thus, a 50%reconstitution rate suggests that there is at least a 5-fold and mostlikely a 15-25 fold expansion in HSCs per transplant. 5 fold expansionis most likely an underestimate since HSCs transplanted in low numberslead to low and variable reconstitution. But our finding that Wnt3Atreated HSCs upon transplantation lead to an average chimerism of 20%(range 12-27%) in the context of a competitive reconstitution suggests agreater than a 5 fold expansion of functional HSCs.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Purified and biologically active Wnt compositions are prepared byexpression of a Wnt protein, preferably a secreted form of a Wntprotein. The protein is then purified in the presence of a detergent tomaintain solubility. A first step is dye ligand chromatography. Thepurified protein fraction can then be further purified by size exclusionchromatography, and by cation exchange chromatography. The methodsprovide for a substantially homogeneous composition of biologicallyactive Wnt protein.

Homogeneous Wnt polypeptide compositions are sufficiently free of otherpeptides or proteins to provide a homogeneous band by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or silverstain. “Substantially homogeneous” protein means a compositioncomprising at least about 90% by weight of the protein, based on totalweight of the composition, preferably at least about 95% by weight, andmay be at least about 99% by weight of protein, based on total weight ofthe composition.

Biologically active Wnt compositions retain the effector functions thatare directly or indirectly caused or performed by native sequence Wntpolypeptides. Effector functions of native sequence Wnt polypeptidesinclude stabilization of β-catenin, stimulation of stem cellself-renewal, C57MG transformation and induction of target genes inXenopus animal cap assays, as well as target gene expression in humanteratocarcinoma cells. The purified Wnt compositions find use in avariety of therapeutic methods, including the maintenance and growth ofstem cells, tissue regeneration, and the like.

For use in the above methods, the invention also provides an article ofmanufacture, comprising: a container, a label on the container, and acomposition comprising an active agent within the container, wherein thecomposition comprises substantially homogeneous biologically active Wntprotein, which is effective in, for example, enhancing proliferationand/or maintenance of stem cells, and the label on the containerindicates that the composition can be used for enhancing proliferationand/or maintenance of those cells.

Definitions

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,and reagents described, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which will be limited only by the appendedclaims.

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the culture” includes reference to one or more culturesand equivalents thereof known to those skilled in the art, and so forth.All technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs unless clearly indicated otherwise.

Wnt protein. Wnt proteins form a family of highly conserved secretedsignaling molecules that regulate cell-to-cell interactions duringembryogenesis. The terms “Wnts” or “Wnt gene product” or “Wntpolypeptide” when used herein encompass native sequence Wntpolypeptides, Wnt polypeptide variants, Wnt polypeptide fragments andchimeric Wnt polypeptides. In some embodiments of the invention, the Wntprotein comprises palmitate covalently bound to a cysteine residue.

A “native sequence” polypeptide is one that has the same amino acidsequence as a Wnt polypeptide derived from nature. Such native sequencepolypeptides can be isolated from cells producing endogenous Wnt proteinor can be produced by recombinant or synthetic means. Thus, a nativesequence polypeptide can have the amino acid sequence of, e.g. naturallyoccurring human polypeptide, murine polypeptide, or polypeptide from anyother mammalian species, or from non-mammalian species, e.g. Drosophila,C. elegans, and the like.

The term “native sequence Wnt polypeptide” includes human and murine Wntpolypeptides. Human wnt proteins include the following: Wnt 1, Genbankreference NP_005421.1; Wnt 2, Genbank reference NP_003382.1, which isexpressed in brain in the thalamus, in fetal and adult lung and inplacenta; two isoforms of Wnt 2B, Genbank references NP_004176.2 andNP_078613.1. Isoform 1 is expressed in adult heart, brain, placenta,lung, prostate, testis, ovary, small intestine and colon. In the adultbrain, it is mainly found in the caudate nucleus, subthalamic nucleusand thalamus. Also detected in fetal brain, lung and kidney. Isoform 2is expressed in fetal brain, fetal lung, fetal kidney, caudate nucleus,testis and cancer cell lines. Wnt 3 and Wnt3A play distinct roles incell-cell signaling during morphogenesis of the developing neural tube,and have the Genbank references NP_110380.1 and X56842. Wnt3A isexpressed in bone marrow. Wnt 4 has the Genbank reference NP_110388.2.Wnt 5A and Wnt 5B have the Genbank references NP_003383.1 and AK013218.Wnt 6 has the Genbank reference NP_006513.1; Wnt 7A is expressed inplacenta, kidney, testis, uterus, fetal lung, and fetal and adult brain,Genbank reference NP_004616.2. Wnt 7B is moderately expressed in fetalbrain, weakly expressed in fetal lung and kidney, and faintly expressedin adult brain, lung and prostate, Genbank reference NP_478679.1. Wnt 8Ahas two alternative transcripts, Genbank references NP_114139.1 andNP_490645.1. Wnt 8B is expressed in the forebrain, and has the Genbankreference NP_003384.1. Wnt 10A has the Genbank reference NP_079492.2.Wnt 10B is detected in most adult tissues, with highest levels in heartand skeletal muscle. It has the Genbank reference NP_003385.2. Wnt 11 isexpressed in fetal lung, kidney, adult heart, liver, skeletal muscle,and pancreas, and has the Genbank reference NP_004617.2. Wnt 14 has theGenbank reference NP_003386.1. Wnt 15 is moderately expressed in fetalkidney and adult kidney, and is also found in brain. It has the Genbankreference NP_003387.1. Wnt 16 has two isoforms, Wnt-16a and Wnt-16b,produced by alternative splicing. Isoform Wnt-16B is expressed inperipheral lymphoid organs such as spleen, appendix, and lymph nodes, inkidney but not in bone marrow. Isoform Wnt-16a is expressed atsignificant levels only in the pancreas. The Genbank references areNP_057171.2 and NP_476509.1.

The term “native sequence Wnt protein” includes the native proteins withor without the initiating N-terminal methionine (Met), and with orwithout the native signal sequence. The native sequence human and murineWnt polypeptides known in the art are from about 348 to about 389 aminoacids long in their unprocessed form reflecting variability(particularly at the poorly conserved amino-terminus and severalinternal sites), contain 21 conserved cysteines, and have the featuresof a secreted protein. The molecular weight of a Wnt polypeptide isabout 38-42 kD.

A “variant” polypeptide means a biologically active polypeptide asdefined below having less than 100% sequence identity with a nativesequence polypeptide. Such variants include polypeptides wherein one ormore amino acid residues are added at the N- or C-terminus of, orwithin, the native sequence; from about one to forty amino acid residuesare deleted, and optionally substituted by one or more amino acidresidues; and derivatives of the above polypeptides, wherein an aminoacid residue has been covalently modified so that the resulting producthas a non-naturally occurring amino acid. Ordinarily, a biologicallyactive Wnt variant will have an amino acid sequence having at leastabout 90% amino acid sequence identity with a native sequence Wntpolypeptide, preferably at least about 95%, more preferably at leastabout 99%.

A “chimeric” Wnt polypeptide is a polypeptide comprising a Wntpolypeptide or portion (e.g., one or more domains) thereof fused orbonded to heterologous polypeptide. The chimeric Wnt polypeptide willgenerally share at least one biological property in common with a nativesequence Wnt polypeptide. Examples of chimeric polypeptides includeimmunoadhesins, combine a portion of the Wnt polypeptide with animmunoglobulin sequence, and epitope tagged polypeptides, which comprisea Wnt polypeptide or portion thereof fused to a “tag polypeptide”. Thetag polypeptide has enough residues to provide an epitope against whichan antibody can be made, yet is short enough such that it does notinterfere with biological activity of the Wnt polypeptide. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 6-60 amino acid residues.

A “functional derivative” of a native sequence Wnt polypeptide is acompound having a qualitative biological property in common with anative sequence Wnt polypeptide. “Functional derivatives” include, butare not limited to, fragments of a native sequence and derivatives of anative sequence Wnt polypeptide and its fragments, provided that theyhave a biological activity in common with a corresponding nativesequence Wnt polypeptide. The term “derivative” encompasses both aminoacid sequence variants of Wnt polypeptide and covalent modificationsthereof.

Biologically Active Wnt. The methods of the present invention providefor substantially homogeneous Wnt compositions that maintain thebiological activity of the starting material. One may determine thespecific activity of a Wnt protein in a composition by determining thelevel of activity in a functional assay, e.g. stabilization ofβ-catenin, promoting growth of stem cells, etc., quantitating the amountof Wnt protein present in a non-functional assay, e.g. immunostaining,ELISA, quantitation on coomasie or silver stained gel, etc., anddetermining the ratio of biologically active Wnt to total Wnt.Generally, the specific activity as thus defined in a substantiallyhomogeneous composition will be at least about 5% that of the startingmaterial, usually at least about 10% that of the starting material, andmay be about 25%, about 50%, about 90% or greater.

Compositions are achieved where the biologically active Wnt protein ispresent at a concentration of at least about 5 μg/ml; usually at leastabout 10 μg/ml, more usually at least about 50 μg/ml, and may be presentat greater than about 100 μg/ml.

Assays for biological activity of Wnt include stabilization ofβ-catenin, which can be measured, for example, by serial dilutions ofthe Wnt composition. As shown in the examples, conditioned medium fromcells expressing Wnt contain about 200 ng Wnt3A/ml, has activitydetectable down to 10-20 fold dilutions in the β-catenin stabilizationassay. Substantially homogeneous Wnt compositions purified from suchconditioned medium contain about 100 μg Wnt3A/ml, and have activitydetectable down to 5000-10000 fold dilutions.

An exemplary assay for Wnt biological activity contacts a Wntcomposition with cells, e.g. mouse L cells. The cells are cultured for aperiod of time sufficient to stabilize b-catenin, usually at least about1 hour, and lysed. The cell lysate is resolved by SDS PAGE, thentransferred to nitrocellulose and probed with antibodies specific forβ-catenin. Other assays include C57MG transformation and induction oftarget genes in Xenopus animal cap assays.

Expression Construct: In the present methods, Wnt may be produced byrecombinant methods. The DNA encoding Wnt polypeptide may be obtainedfrom any cDNA library prepared from tissue expressing the Wntpolypeptide mRNA, prepared from various sources according to the desiredWnt. The Wnt polypeptide-encoding gene may also be obtained from agenomic library or by oligonucleotide synthesis. As described above,there are many Wnt polypeptides and genetic sequences known in the art.Libraries may be screened with probes (such as antibodies to the Wntpolypeptide, or oligonucleotides of about 20-80 bases) designed toidentify the gene of interest or the protein encoded by it. Screeningthe cDNA or genomic library with the selected probe may be conductedusing standard procedures as described in Sambrook et at, MolecularCloning: A Laboratory Manual (New York: Cold Spring Harbor LaboratoryPress, 1989). An alternative means to isolate the gene encoding Wntpolypeptide is to use PCR methodology.

Amino acid sequence variants of Wnt polypeptide are prepared byintroducing appropriate nucleotide changes into the Wnt polypeptide DNA,or by synthesis of the desired Wnt polypeptide. Such variants representinsertions, substitutions, and/or specified deletions of, residueswithin or at one or both of the ends of the amino acid sequence of anaturally occurring Wnt polypeptide. Preferably, these variantsrepresent insertions and/or substitutions within or at one or both endsof the mature sequence, and/or insertions, substitutions and/orspecified deletions within or at one or both of the ends of the signalsequence of the Wnt polypeptide. Any combination of insertion,substitution, and/or specified deletion is made to arrive at the finalconstruct, provided that the final construct possesses the desiredbiological activity as defined herein. The amino acid changes also mayalter post-translational processes of the Wnt polypeptide, such aschanging the number or position of glycosylation sites, altering themembrane anchoring characteristics, and/or altering the intracellularlocation of the Wnt polypeptide by inserting, deleting, or otherwiseaffecting the leader sequence of the Wnt polypeptide.

The nucleic acid (e.g., cDNA or genomic DNA) encoding the Wntpolypeptide is inserted into a replicable vector for expression. Manysuch vectors are available. The vector components generally include, butare not limited to, one or more of the following: an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence.

Wnt polypeptides may be produced recombinantly not only directly, butalso as a fusion polypeptide with a heterologous polypeptide, e.g. asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be a part ofthe Wnt polypeptide DNA that is inserted into the vector. Theheterologous signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. In mammalian cell expression the native signal sequence maybe used, or other mammalian signal sequences may be suitable, such assignal sequences from other animal Wnt polypeptide, and signal sequencesfrom secreted polypeptides of the same or related species, as well asviral secretory leaders, for example, the herpes simplex gD signal.

Expression vectors usually contain a selection gene, also termed aselectable marker. This gene encodes a protein necessary for thesurvival or growth of transformed host cells grown in a selectiveculture medium. Host cells not transformed with the vector containingthe selection gene will not survive in the culture medium. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli.

Expression vectors will contain a promoter that is recognized by thehost organism and is operably linked to the Wnt coding sequence.Promoters are untranslated sequences located upstream (5′) to the startcodon of a structural gene (generally within about 100 to 1000 bp) thatcontrol the transcription and translation of particular nucleic acidsequence to which they are operably linked. Such promoters typicallyfall into two classes, inducible and constitutive. Inducible promotersare promoters that initiate increased levels of transcription from DNAunder their control in response to some change in culture conditions,e.g., the presence or absence of a nutrient or a change in temperature.A large number of promoters recognized by a variety of potential hostcells are well known. Both a native Wnt polypeptide promoter sequenceand many heterologous promoters may be used to direct expression of aWnt polypeptide. However, heterologous promoters are preferred, as theygenerally permit greater transcription and higher yields.

Promoters suitable for use with prokaryotic hosts include theβ-lactamase and lactose promoter systems, alkaline phosphatase, atryptophan (trp) promoter system, and hybrid promoters such as the tacpromoter. However, other known bacterial promoters are also suitable.Such nucleotide sequences have been published, thereby enabling askilled worker operably to ligate them to a DNA coding sequence.Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the coding sequence.

Promoter sequences are known for eukaryotes. Examples of suitablepromoting sequences for use with yeast hosts include the promoters for3-phosphoglyceratekinase or other glycolytic enzymes, such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Other yeast promoters, whichare inducible promoters having the additional advantage of transcriptioncontrolled by growth conditions, are the promoter regions for alcoholdehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymesassociated with nitrogen metabolism, metallothionein,glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible formaltose and galactose utilization. Suitable vectors and promoters foruse in yeast expression are further described in EP 73,657. Yeastenhancers also are advantageously used with yeast promoters.

Transcription from vectors in mammalian host cells may be controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovinepapilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus,hepatitis-B virus and most preferably Simian Virus 40 (SV40), fromheterologous mammalian promoters, e.g., the actin promoter, PGK(phosphoglycerate kinase), or an immunoglobulin promoter, fromheat-shock promoters, provided such promoters are compatible with thehost cell systems. The early and late promoters of the SV40 virus areconveniently obtained as an SV40 restriction fragment that also containsthe SV40 viral origin of replication. The immediate early promoter ofthe human cytomegalovirus is conveniently obtained as a HINDIII Erestriction fragment.

Transcription by higher eukaryotes is often increased by inserting anenhancer sequence into the vector. Enhancers are cis-acting elements ofDNA, usually about from 10 to 300 bp, which act on a promoter toincrease its transcription. Enhancers are relatively orientation andposition independent, having been found 5′ and 3′ to the transcriptionunit, within an intron, as well as within the coding sequence itself.Many enhancer sequences are now known from mammalian genes (globin,elastase, albumin, α-fetoprotein, and insulin). Typically, however, onewill use an enhancer from a eukaryotic cell virus. Examples include theSV40 enhancer on the late side of the replication origin, thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the expression vector at a position 5′ or3′ to the coding sequence, but is preferably located at a site 5′ fromthe promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding Wnt polypeptide.

Construction of suitable vectors containing one or more of theabove-listed components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and re-ligated in theform desired to generate the plasmids required. For analysis to confirmcorrect sequences in plasmids constructed, the ligation mixtures areused to transform host cells, and successful transformants selected byampicillin or tetracycline resistance where appropriate. Plasmids fromthe transformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced.

Particularly useful in the practice of this invention are expressionvectors that provide for the transient expression in mammalian cells. Ingeneral, transient expression involves the use of an expression vectorthat is able to replicate efficiently in a host cell, such that the hostcell accumulates many copies of the expression vector and, in turn,synthesizes high levels of a desired polypeptide encoded by theexpression vector. Transient expression systems, comprising a suitableexpression vector and a host cell, allow for the convenient positiveidentification of polypeptides encoded by cloned DNAs, as well as forthe rapid screening of such polypeptides for desired biological orphysiological properties.

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis, Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable expression hosts. Saccharomyces cerevisiae,or common baker's yeast, is the most commonly used among lowereukaryotic host microorganisms. However, a number of other genera,species, and strains are commonly available and useful herein, such asSchizosaccharomyces pombe; Kluyveromyces hosts such as K. lactis, K.fragilis, etc.; Pichia pastoris; Candida; Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as Penicillium, Tolypocladium, and Aspergillus hosts such asA. nidulan, and A. niger.

Suitable host cells for the expression of glycosylated polypeptide maybe derived from multicellular organisms. Such host cells are capable ofcomplex processing and glycosylation activities. In principle, anyhigher eukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. Examples of invertebrate cells include plant andinsect cells. Numerous baculoviral strains and variants andcorresponding permissive insect host cells from hosts such as Spodopterafrugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus(mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori havebeen identified. A variety of viral strains for transfection arepublicly available, e.g., the L-1 variant of Autographa California NPVand the Bm-5 strain of Bombyx mori NPV, and such viruses may be used asthe virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens. During such incubation of the plant cellculture, the DNA coding sequence is transferred to the plant cell hostsuch that it is transfected, and will, under appropriate conditions,express the DNA. In addition, regulatory and signal sequences compatiblewith plant cells are available, such as the nopaline synthase promoterand polyadenylation signal sequences.

In a preferred embodiment, Wnt proteins are produced in vertebratecells, and more particularly in vertebrate cells that, upon expressionof a Wnt protein, post-translationally modify the protein by covalentattachment of a lipid, e.g. palmitate, to a conserved cysteine residue.For example, in murine Wnt3A, cys 77 is palmitoylated. This cysteineresidue is conserved in all Wnt proteins. Other Wnt proteins isolated bythe methods of the invention include, inter alia, DWnt8, mouse Wnt5A,and Drosophila Wingless.

The ability of a cell to palmitoylate Wnt may be empirically determinedby synthesis of the Wnt protein in the presence of labeled palmitate,and determining the incorporation of such a label into the Wnt product.Protein products of the Drosophila porcupine and homologs thereof in,for example, mammalian cells, may catalyze such acylation of Wntproteins (see, for example, Hofmann (2000) Trends Biochem. Sci.25:111-112; Tanaka et al. (2002) J. Biol. Chem. 277:12816-12823, hereinincorporated by reference).

Examples of useful mammalian host cell lines are mouse L cells(L-M[TK-], ATCC#CRL-2648), monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture; baby hamster kidney cells(BHK, ATCC CCL 10); Chinese hamster ovary cells/DHFR (CHO); mousesertoli cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); Africangreen monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervicalcarcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells(W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammarytumor (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and ahuman hepatoma line (Hep G2).

Host cells are transfected with the above-described expression vectorsfor Wnt polypeptide production, and cultured in conventional nutrientmedia modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.Mammalian host cells may be cultured in a variety of media. Commerciallyavailable media such as Ham's F10 (Sigma), Minimal Essential Medium((MEM), Sigma), RPMI 1640 (Sigma), and Dulbecco's Modified Eagle'sMedium ((DMEM), Sigma) are suitable for culturing the host cells. Any ofthese media may be supplemented as necessary with hormones and/or othergrowth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleosides (such as adenosine andthymidine), antibiotics, trace elements, and glucose or an equivalentenergy source. Any other necessary supplements may also be included atappropriate concentrations that would be known to those skilled in theart. The culture conditions, such as temperature, pH and the like, arethose previously used with the host cell selected for expression, andwill be apparent to the ordinarily skilled artisan.

Nucleic acids are “operably linked” when placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for asignal sequence is operably linked to DNA for a polypeptide if it isexpressed as a preprotein that participates in the secretion of thepolypeptide; a promoter or enhancer is operably linked to a codingsequence if it affects the transcription of the sequence; or a ribosomebinding site is operably linked to a coding sequence if it is positionedso as to facilitate translation. Generally, “operably linked” means thatthe DNA sequences being linked are contiguous, and, in the case of asecretory leader, contiguous and in reading phase. However, enhancers donot have to be contiguous. Linking is accomplished by ligation atconvenient restriction sites. If such sites do not exist, the syntheticoligonucleotide adapters or linkers are used in accordance withconventional practice.

Purification of Wnt Protein

Wnt proteins are preferably recovered from the culture medium as asecreted polypeptide, although it can also be recovered from host celllysates. Wnt proteins have been found to be unexpectedly hydrophobic,due to the lipid modification. As such, the protein is preferablypurified in the presence of a detergent to maintain solubility. Suitabledetergents for this purpose include non-anionic detergents, andzwitterionic detergents, which may be used at a concentration of fromabout 0.25% to about 2.5%, usually at a concentration of from about 0.5%to 1.5%, and preferably at a concentration of about 1%.

Non-anionic detergents include the Triton™ family of detergents, e.g.Triton™ X-15; Triton™ X-35; Triton™ X-45; Triton™ X-100; Triton™ X-102;Triton™ X-114; Triton™ X-165, etc. All of these heterogeneous detergentshave a branched 8-carbon chain attached to an aromatic ring. Thisportion of the molecule contributes most of the hydrophobic nature ofthe detergent. Triton™ X-100 and NP-40 are very similar in structure andhydrophobicity and are interchangeable in most applications includingcell lysis. Brij™ detergents are also similar in structure to Triton™ Xdetergents in that they have varying lengths of polyoxyethylene chainsattached to a hydrophobic chain. However, unlike Triton™ X detergents,the Brij™ detergents do not have an aromatic ring and the length of thecarbon chains can vary. Brij™ 58 is most similar to Triton™ X 100 in itshydrophobic/hydrophilic characteristics. The Tween™ detergents arenondenaturing, nonionic detergents, which are polyoxyethylene sorbitanesters of fatty acids. Tween™ 80 is derived from oleic acid with a C₁₈chain while Tween™ 20 is derived from lauric acid with a C₁₂ chain.

The zwitterionic detergent, CHAPS, is a sulfobetaine derivative ofcholic acid. This zwitterionic detergent is useful for membrane proteinsolubilization when protein activity is important. This detergent isuseful over a wide range of pH (pH 2-12) and is easily removed fromsolution by dialysis due to high CMCs (8-10 mM). A preferred non-ionicdetergent is Triton-X 100 or NP-40.

A protease inhibitor, such as phenyl methyl sulfonyl fluoride (PMSF)also may be useful to inhibit proteolytic degradation duringpurification, and antibiotics may be included to prevent the growth ofadventitious contaminants.

A first separation step is an affinity chromatography step. Affinitychromatography makes use of the highly specific binding sites usuallypresent in biological macromolecules, separating molecules on theirability to bind a particular ligand. Covalent bonds attach the ligand toan insoluble, porous support medium in a manner that overtly presentsthe ligand to the protein sample, thereby using natural biospecificbinding of one molecular species to separate and purify a second speciesfrom a mixture. Antibodies are commonly used in affinity chromatography.

Preferably a microsphere or matrix is used as the support for affinitychromatography. Such supports are known in the art and commerciallyavailable, and include activated supports that can be coupled to thelinker molecules. For example, Affi-Gel supports, based on agarose orpolyacrylamide are low pressure gels suitable for most laboratory-scalepurifications with a peristaltic pump or gravity flow elution. Affi-Prepsupports, based on a pressure-stable macroporous polymer, are suitablefor preparative and process scale applications.

In a preferred embodiment, the affinity chromatography step utilizesdye-ligand chromatography, in which synthetic textile dyes are used inlieu of natural substrates, cofactors or effectors commonly employed asimmobilized ligands. Most work with affinity chromatography has beendone with the Cibacron Blue 3GA ligand. The Matrex Blue A ligand is aslight variation of Cibacron Blue 3GA. Matrex Blue A is coupled directlyto an agarose support through the triazine ring by CNBr. Wnt may beeluted from the affinity column or resin in a buffer with increasedionicity.

The Wnt polypeptide composition may then be size selected, for exampleby gel filtration. Gel filtration chromatography (also known assize-exclusion chromatography or molecular sieve chromatography) is usedto separate proteins according to their size. In gel filtration, aprotein solution is passed through a column that is packed withsemipermeable porous resin. The semipermeable resin has a range of poresizes that determines the size of proteins that can be separated withthe column. While the pore size may vary with the exact Wnt protein ofinterest, typically a pore in the range of 100-300 will be used.Examples of suitable resins include Bio-Gel P-300, Sephadex G-200,Superdex 200, and the like.

The column must first be equilibrated with the desired buffer, which asdescribed above will contain a suitable detergent. This is accomplishedby simply passing several column volumes of the buffer through thecolumn. Equilibration is an important step because the equilibrationbuffer is the buffer in which the protein sample will elute. Next, thesample is loaded onto the column and allowed to enter the resin. Thenmore of the equilibration buffer is passed through the column toseparate the sample and elute it from the column. Fractions arecollected as the sample elutes from the column.

The Wnt protein may be further separated by cation exchangechromatography, for example using heparin. This cation is a sulfatedglucosaminoglycan which can be extracted from the native proteoglycan.Heparin consists of alternating units of uronic acid and D-glucosamine,most of which are substituted with one or two sulfate groups. Themolecular weight of the polymer is distributed over the range 5000-30000. Heparin is covalently coupled to highly cross-linked resin.

The final Wnt composition may be concentrated, filtered, dialyzed, etc.,using methods known in the art. For therapeutic applications, the Wntpolypeptides are administered to a mammal, preferably a human, in aphysiologically acceptable dosage form, including those that may beadministered to a human intravenously as a bolus or by continuousinfusion over a period of time. Alternative routes of administrationinclude intramuscular, intraperitoneal, intra-cerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. The Wnt polypeptides also are suitablyadministered by intratumoral, peritumoral, intralesional, orperilesional routes or to the lymph, to exert local as well as systemictherapeutic effects.

Such dosage forms encompass physiologically acceptable carriers that areinherently non-toxic and non-therapeutic. Examples of such carriersinclude ion exchangers, alumina, aluminum stearate, lecithin, serumproteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts, orelectrolytes such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, and PEG. Carriers for topical or gel-based forms of Wntpolypeptides include polysaccharides such as sodiumcarboxymethylcellulose or methylcellulose, polyvinylpyrrolidone,polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, PEG, andwood wax alcohols. For all administrations, conventional depot forms aresuitably used. Such forms include, for example, microcapsules,nano-capsules, liposomes, plasters, inhalation forms, nose sprays,sublingual tablets, and sustained-release preparations. The Wntpolypeptide will typically be formulated in such vehicles at aconcentration of about 0.1 μg/ml to 100 μg/ml.

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the conditionsdescribed above is provided. The article of manufacture comprises acontainer and a label. Suitable containers include, for example,bottles, vials, syringes, and test tubes. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition that is effective for treating the condition and mayhave a sterile access port (for example the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). The active agent in the composition is theWnt protein. The label on, or associated with, the container indicatesthat the composition is used for treating the condition of choice.Further container(s) may be provided with the article of manufacturewhich may hold, for example, a pharmaceutically-acceptable buffer, suchas phosphate-buffered saline, Ringer's solution or dextrose solution.The article of manufacture may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, syringes, and package inserts withinstructions for use.

In one embodiment of the invention the Wnt protein composition is usedto enhance the proliferation and/or self-renewal of stem cells in vivoor in vitro. The term stem cell is used herein to refer to a mammaliancell that has the ability both to self-renew, and to generatedifferentiated progeny (see Morrison et al. (1997) Cell 88:287-298).Generally, stem cells also have one or more of the following properties:an ability to undergo asynchronous, or symmetric replication, that iswhere the two daughter cells after division can have differentphenotypes; extensive self-renewal capacity; capacity for existence in amitotically quiescent form; and clonal regeneration of all the tissue inwhich they exist, for example the ability of hematopoietic stem cells toreconstitute all hematopoietic lineages. “Progenitor cells” differ fromstem cells in that they typically do not have the extensive self-renewalcapacity, and often can only regenerate a subset of the lineages in thetissue from which they derive, for example only lymphoid, or erythroidlineages in a hematopoietic setting.

Stem cells may be characterized by both the presence of markersassociated with specific epitopes identified by antibodies and theabsence of certain markers as identified by the lack of binding ofspecific antibodies. Stem cells may also be identified by functionalassays both in vitro and in vivo, particularly assays relating to theability of stem cells to give rise to multiple differentiated progeny.

Stem cells of interest include hematopoietic stem cells and progenitorcells derived therefrom (U.S. Pat. No. 5,061,620); neural crest stemcells (see Morrison et al. (1999) Cell 96:737-749); embryonic stemcells; mesenchymal stem cells; mesodermal stem cells; etc.

For in vitro use, a population of cells comprising progenitor and/orstem cells is cultured in vitro in the presence of biologically activeWnt sufficient to maintain or increase the number of assayableprogenitor cells in the culture. The number of assayable progenitorcells may be demonstrated by a number of assays. After one week theprogenitor cell cloning efficiency will usually be at least about 75%that of the starting cell population, more usually 100% that of thestarting cell population, and may be as high as 200% that of thestarting cell population.

These cells may find various applications for a wide variety ofpurposes. The cell populations may be used for screening variousadditives for their effect on growth and the mature differentiation ofthe cells. In this manner, compounds which are complementary, agonistic,antagonistic or inactive may be screened, determining the effect of thecompound in relationship with one or more of the different cytokines.

The populations may be employed as grafts for transplantation. Forexample, hematopoietic cells are used to treat malignancies, bone marrowfailure states and congenital metabolic, immunologic and hematologicdisorders. Marrow samples may be taken from patients with cancer, andenriched populations of hematopoietic stem cells isolated by means ofdensity centrifugation, counterflow centrifugal elutriation, monoclonalantibody labeling and fluorescence activated cell sorting. The stemcells in this cell population are then expanded in vitro and can serveas a graft for autologous marrow transplantation. The graft will beinfused after the patient has received curative chemo-radiotherapy.

The cells of interest are typically mammalian, where the term refers toany animal classified as a mammal, including humans, domestic and farmanimals, and zoo, laboratory, sports, or pet animals, such as dogs,horses, cats, cows, mice, rats, rabbits, etc. Preferably, the mammal ishuman.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

The present invention has been described in terms of particularembodiments found or proposed by the present inventor to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. For example, due to codon redundancy, changescan be made in the underlying DNA sequence without affecting the proteinsequence. Moreover, due to biological functional equivalencyconsiderations, changes can be made in protein structure withoutaffecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

EXPERIMENTAL

Active Wnt molecules, including the product of the mouse Wnt3A gene,were isolated. By mass spectrometry, the proteins were found to bepalmitoylated on a conserved cysteine. Enzymatic removal of thepalmitate, or site-directed and natural mutations of the modifiedcysteine, result in loss of activity, indicating that the lipid isimportant for signaling. The purified Wnt3A protein induces self-renewalof hematopoietic stem cells, signifying its potential use in tissueengineering.

We expressed several Wnt genes, including Wnt3A, in a variety of celllines and generated antibodies to monitor Wnt protein secretion into themedium. For purification purposes, we selected clones of cells secretinghighest amounts of protein (200 ng/ml for Wnt3A from mouse L cells). Wetested the activity of Wnt3A by assaying its ability to stabilizecytosolic β-catenin, a known target and signal transduction component ofWnt signaling. Mouse L cells accumulate high levels of β-catenin proteinafter a two hour incubation with Wnt3A conditioned medium (FIG. 1B, toppanel). After initial characterization of secreted Wnt3A indicated thatit is hydrophobic (see below), we designed a purification protocol thatstarts with chromatography on Blue (Cibacron Blue 3GA) Sepharose in thepresence of the detergent CHAPS. Under these conditions, Wnt3A bindswith high selectivity to the resin and can be eluted in a relativelypure form by increasing ionic strength (FIG. 1A; Table 1). Approximately60% of added Wnt3A is recovered in this step with a nearly 2500-foldenrichment. We then separated Wnt-containing fractions by size exclusionchromatography on a Superdex 200 column, and finally by cation exchangeon heparin (Table 1). These steps yielded fractions of Wnt3A that weregreater than 95% pure as assessed by Coomassie staining (FIG. 1A). Bysize exclusion chromatography, we determined that active Wnt3A ismonomeric.

We have successfully applied similar purification methods to a varietyof other Wnts, including DWnt8 (FIG. 1A), mouse Wnt5A and DrosophilaWingless. Throughout the purification, we measured the ability of Wnt3Ato stabilize β-catenin in L cells. The final purified product exhibitedno loss in activity compared to the original starting material (FIG.1B). The purified Wnt3A protein retains the range of activities expectedfor a Wnt protein. For example, we tested the effect of Wnt3A protein onXenopus animal cap explants and found that two known target genes,Siamois and Xnr3 are induced by Wnt3A (FIG. 1C). As a further assay forWnt activity, we used C57MG cells, a line derived from the mouse mammarygland that can be morphologically transformed by Wnt gene expression.Purified Wnt3A promotes the morphological transformation of these cells(FIG. 1D) similar to that of Wnt gene transfection. In addition, theprotein can induce expression of known transcriptional Wnt targetsincluding MSX1, CYCLIN D1, and MYC in human teratocarcinoma cells.

Wnt proteins are modified by palmitoylation on a conserved cysteine. Allpurification steps required the presence of detergent to maintainsolubility and activity, suggesting that Wnt proteins are hydrophobic.We used the two-phase separation property of the detergent Triton X-114to test this. The majority of Wnt3A partitioned to the detergent phase(FIG. 2A), a behavior characteristic of highly hydrophobic proteins suchas integral membrane proteins. Since the primary amino acid sequence ofsecreted Wnt does not contain long stretches of hydrophobic residues, weused metabolic labeling to test whether Wnt is post-translationallymodified by lipid attachment. We found that the protein is labeled withtritiated palmitate (FIG. 2B). Evidence for the functional importance ofthe lipid modification came from treatment of Wnt3A with Acyl-ProteinThioesterase-1 (APT-1), an enzyme which removes palmitate from Gproteins and other thioacyl protein substrates. This treatment shiftsWnt3A to the water phase in the Triton X-114 phase separation experiment(FIG. 2C), suggesting that APT-1 removes a thioester linked lipid, suchas palmitate. APT-1 also blocks Wnt3A's ability to stabilize β-catenin(FIG. 2C).

In order to map the lipid attachment site on the Wnt polypeptide wesubjected proteolytic peptide fragments of both Wnt3A and DWnt8 toliquid chromatography tandem mass spectrometry (LC-MS/MS), whichidentifies molecular masses of the ionized peptides and obtains primaryamino acid sequence information through collision induced fragmentation.In both proteins we identified ions whose masses were consistent withthe addition of 238 daltons (the mass of palmitate is 256 accounting forthe loss of water in the formation of a thioester linkage) and whichproduced fragmentation data consistent with a peptide containing aconserved cysteine modified by palmitate (C77 in Wnt3A and C51 in DWnt8;underlined in FIG. 2D). This cysteine is absolutely conserved among allWnt family members (bold in FIG. 2D); it is the most amino-terminalconserved cysteine of the Wnt family.

To test for requirement of C77 in cell culture, we mutated it to alaninein Wnt3A and expressed the mutant protein (C77>A; FIG. 2E) in 293 and inL cells. The mutant Wnt3A protein was secreted at levels similar to thatof the wild-type protein. This indicated that the mutation, unlike manyother cysteine mutations in Wnts, does not interfere with the folding ofthe protein. However, when the Wnt3A (C77>A) protein was subjected tothe Triton X-114 phase separation test, it partitioned in the waterphase, indicating that it had lost its hydrophobic character (FIG. 2E).In a β-catenin assay on L cells, Wnt3A(C77>A) was not active over arange of concentrations tested (FIG. 2E, left). In a transfection assayon 293 cells however, there was a noticeable increase in theintracellular levels of β-catenin, demonstrating that the Wnt3A(C77>A)mutant retains some activity when expressed at high levels in anautocrine manner (FIG. 2E, right).

Interestingly, a natural loss-of-function allele of the C. elegansegl-20 gene (egl-20 N585) contains a serine replacing the cysteinecorresponding to C77 (FIG. 2D). Moreover, in a survey of wingless (wg)alleles in Drosophila, we found that the wg S21 allele contains atyrosine instead of that same cysteine (FIG. 2D). Thus, our data areconsistent with the lipid modification being important for Wnt signalingactivity.

Purified Wnt3A causes self-renewal of hematopoietic stem cells (HSC) invitro. To test directly whether Wnt3A can be used as a reagent tocontrol cell fate in a well-characterized stem cell system, we appliedthe isolated protein to purified hematopoietic stem cells (HSCs). SingleHSCs responded well to the Wnt3A protein in the presence of limitingdoses of Steel factor. Over a period of 7 days, the frequency of cellsproliferating was 5.8 fold greater compared to control conditions (FIGS.3A and B). The majority of cells (82%) were undifferentiated in thatthey did not express markers for differentiated lineages. 30 percent ofthe lineage negative cells expressed c-kit and Sca-1, consistent with anHSC phenotype, while 64% were at the stage of myeloid progenitors(c-kit⁺ Sca-1⁻; FIG. 3C, D). In contrast, incubation of HSCs withunfractionated Wnt3A-conditioned medium, in which Wnt3A itself ispresent at a similar concentration, resulted in a significant fraction(86%) of the cells expressing markers specific for differentiatedlineages (FIG. 3C). This suggests that conditioned medium containsfactors not present in purified Wnt3A that promote differentiation,underscoring the importance of having purified Wnt proteins availablefor the purpose of maintaining the self-renewing fate of HSCs.

To determine whether the cells that proliferated in response to Wnt3Atruly maintained HSC activity, we carried out transplantation analysis.Single HSCs were plated in Terasaki plates, and treated with Wnt3A orcontrol media for a period of six days. In previous experiments weshowed that culturing cells with Steel factor alone (our controlconditions) while inducing proliferation, does not induce self-renewalin vitro. Each well containing cells that responded to Wnt3A from asingle cell was separately injected into lethally irradiated mice, andanalyzed after 6 weeks of reconstitution (FIG. 3E). If no self-renewalhad occurred, only 10% of the mice would be expected to be reconstitutedsuccessfully (see Figure Legend). In contrast 100% of the transplantedmice contained donor-derived cells (FIG. 3F), suggesting that HSCs hadundergone self-renewal in response to purified Wnt3A. Furthermore, allthree B, T and myeloid lineages were generated in 50% of the transplantrecipients.

In conclusion, we have established methods to purify significantquantities of pure and active Wnt proteins, which can be used to in Wntmediated biological activities, including the mediating the self-renewalof HSCs and potentially other stem cells. We found that Wnts areunexpectedly hydrophobic and post-translationally modified bypalmitoylation, a property that explains the poor solubility of theproteins. It is interesting to note that the protein products of theDrosophila porcupine and C. elegans mom-1 genes have homology with acyltransferases and may catalyze Wnt acylation. Moreover, the Porcupineprotein can bind to a domain in Wingless encompassing the acylation siteporcupine and mom-1 have phenotypes similar to Wnt alleles and arerequired in Wnt producing cells, indicating that the lipid is anintegral part of signaling activity. However, overexpression of Winglessin the Drosophila embryo can overcome the absence of porcupine, just ashigh expression of Wnt3A(C77>A) can lead to a modest increase inβ-catenin (FIG. 2D). This suggests that the lipid functions to increasethe local concentration of Wnt on membranes, and that its absence can beovercome by high levels of expression. While palmitoylation of secretedproteins appears unusual, there is an intriguing parallel between Wntand Hedgehog signaling, as the Hedgehog protein is also palmitoylated.

Methods

Purification of Wnt3A. Mouse L cells (L-M[TK-], ATCC#CRL-2648) werecultured in DMEM, 10% fetal bovine serum (FBS) and antibiotics. Thesecells were stably transfected with a vector containing the Wnt3A cDNAunder the control of the PGK promoter, and G418 resistant clones wereselected and screened for production of Wnt3A protein (ATCC#CRL-2647).Drosophila S2 cells were used to produce the DWnt8 protein, which wasexpressed from a heat-shock promoter. Two liters of 0.2 μm filteredmedium from L-Wnt3A cells, conditioned for four days, was adjusted to 1%Triton X-100, filtered and applied to Blue (Cibacron Blue) Sepharose HP(Amersham Biosciences) column (bed volume of 120 ml) which waspreviously equilibrated in binding buffer (150 mM KCl, 20 mM Tris-HCl,1% CHAPS, pH7.5). The column was then washed with 4 column volumes ofbinding buffer.

Bound proteins were eluted with a single step to 1.5M KCl, 20 mMTris-HCl, 1% CHAPS, pH7.5. Wnt3A eluted in two pools, each of whichcontained similar amounts of Wnt3A protein; however, the second poolcontained significantly less total protein than the first (30.6 mg totalprotein in the first pool and 2.16 mg in the second pool). Fractionsfrom this second pool were combined, concentrated to 12.5 ml on aCentricon 30 ultrafiltration device (Amicon), and fractionated on aHiLoad 26/60 Superdex 200 column (Amersham Biosciences) in 1× phosphatebuffered saline (PBS), 1% CHAPS, pH7.3. Wnt3A containing fractions werethen fractionated on a 1 ml HiTrap Heparin column (Amersham Biosciences)in a single step elution from 1×PBS, 1% CHAPS to 1×PBS, 1% CHAPS, 1MNaCl. N-terminal sequence of 1 μg purified Wnt3A was obtained byautomated Edman degradation on a Procise 494 ABI sequenator. IsolatedWnt3A begins with residue 19 of the predicted amino acid sequence(SYPIVWVSLAVGPQYS) indicating that the protein is proteolyticallyprocessed to remove the signal sequence.

Triton X-114 Phase Separation. Wnt3A conditioned medium was mixed 1:1with ice cold 4.5% Triton X-114, 150 mM NaCl, 10 mM Tris-HCl, pH7.5,incubated on ice for 5 minutes, then at 31° C. for 5 minutes, andcentrifuged at 2000×g at 31° C. for 5 minutes. The top, aqueous phasewas separated from the bottom Triton X-114 phase and equal volumes wereimmuno-blotted with the anti-Wnt3A antibody.

In Vivo Labeling of Wnt3A with Palmitate. L and L-Wnt3A cells werecultured in 10 cm plates for three days after a 1:10 split. [9,10(n)⁻³H] Palmitic acid (Amersham Biosciences) was added to the medium at aconcentration of 0.1 mCi/ml and incubated for 5 hours at 37° C. Themedia were filtered, CHAPS was added to a concentration of 1%, and thenre-filtered. The individual media were fractionated on 1 ml HiTrap BlueSepharose columns (Amersham Biosciences) as described above. The Wnt3Acontaining fractions or analogous fractions were precipitated with TCAand analyzed by gel-electrophoresis and autoradiography.

Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). PurifiedWnt3A and DWnt8 were precipitated with trichloroacetic acid,re-suspended, alkylated and reduced as described by Wu & Nusse (2002) JBiol Chem 277:41762-41769. The sample was split into 3 aliquots,digested separately with trypsin, subtilisin and elastase, and theresulting peptide mixtures were recombined and analyzed by MudPIT asdescribed in Washburn et al. (2001) Nat Biotechnol 19:242-7 withmodifications as described by MacCoss et al. (2002) Proc Natl Acad SciUSA 99:7900-5, on a Finnigan LCQ-Deca. Tandem mass spectra were searchedagainst a database of predicted open reading frames to which commoncontaminants such as keratin and trypsin were added. Search results werefiltered and grouped using the DTASelect program and identificationsconfirmed through manual evaluation of spectra. The data weresubsequently searched with a differential modification on Cysteine of238 to identify sites of palmitoylation. We also observed this peptidein its unpalmitoylated form. The lipid may be labile and lost during themanipulation of the sample, or there may be a pool of unmodified Wnt-3Apresent in the preparation. We found the following masses [(M+H)+]:Wnt3a peptide unmodified: 1374.51 (predicted: 1374.465); Wnt3A peptidemodified: 1556.10 (predicted: 1555.465); DWnt8 peptide unmodified:1583.37 (predicted: 1583.667); DWnt8 peptide modified: 1764.23(predicted: 1764.667). Even though the MS/MS analysis of Wnt3A and DWnt8identified 85% and 90% of the primary amino acid sequences,respectively, we did not obtain evidence for additional lipidmodifications on other residues (S, T, Y, K, R).

Acyl-Protein Thioesterase Treatment of Wnt3A. 100 ng Wnt3A was treatedin the presence of 1 μg BSA with 1, 10, 100 or 1000 ng APT-1 in buffer(1×PBS, 1% CHAPS, 1M NaCl) in a total volume of 10 μl and incubatedovernight at 30° C. The reaction products were analyzed in the β-cateninstabilization assay on L cells and in the Triton X-114 phase separationassay.

HSC Isolation and Assays. HSCs were sorted from mouse bone marrow ofBcl-2 transgenic mice using antibodies. Cells were sorted on expressionof c-kit, Sca-1, low levels of Thy1.1, and low to negative levels oflineage markers (Lin) using clonecyte software and the single celldeposition unit (Becton Dickinson Immunocytometry systems).

TABLE 1 Purification Table Protein Wnt3A concen- Total concen- Wnt3AVolume tration protein tration (μg) Wnt3A 2 L 4.46 mg/ml 8920 mg 200ng/ml 400 μg CM Blue 60 ml 36.0 μg/ml 2.16 mg 4 μg/ml 240 μg SepharoseGel 36 ml 17.1 μg/ml 615 μg 5 μg/ml 180 μg filtration Heparin 1.15 ml104 μg/ml 120 μg 100 μg/ml 11 μg cation exchange The concentration ofWnt3A protein in the conditioned medium was determined by comparing itssignal intensity on a Wnt3A inmmunoblot to that of a serial dilution ofa known amount of a purified Wnt3A protein.

The concentration of Wnt3A protein in the conditioned medium wasdetermined by comparing its signal intensity on a Wnt3A immuno-blot tothat of a serial dilution of a known amount of purified Wnt3A protein.

What is claimed is:
 1. An article of manufacture, comprising: acontainer; a label on the container; and a composition comprising abiologically active Wnt protein comprising a lipid moiety within thecontainer.
 2. The article of manufacture of claim 1, wherein thecomposition further comprises a liposome.
 3. The article of manufactureof claim 1, wherein the biologically active Wnt protein is a human Wntprotein.
 4. The article of manufacture of claim 3, wherein the human Wntprotein is human Wnt3A protein.
 5. The article of manufacture of claim1, wherein specific activity of the biologically active Wnt protein isat least 5% of the specific activity of Wnt protein in a lysate orculture medium.
 6. The article of manufacture of claim 1, wherein thebiologically active Wnt protein is at a concentration of at least about10 μg/mL.
 7. The article of manufacture of claim 1, wherein thebiologically active Wnt protein is formulated for intravenous,intramuscular, intraperitoneal, intracerobrospinal, subcutaneous,intra-articular, intrasynovial, intrathecal, oral, topical, orinhalation administration.
 8. The article of manufacture of claim 1,wherein the composition further comprises isolated cells.
 9. The articleof manufacture of claim 8, wherein the isolated cells are stem cells.10. The article of manufacture of claim 9, wherein the stem cells arehematopoietic stem cells.
 11. The article of manufacture of claim 1,wherein the container is glass.
 12. The article of manufacture of claim1, wherein the container is plastic.
 13. The article of manufacture ofclaim 1, further comprising a pharmaceutically-acceptable buffer. 14.The article of manufacture of claim 1, further comprising a diluent. 15.The article of manufacture of claim 1, further comprising needles andsyringes.
 16. The article of manufacture of claim 1, further comprisingpackage inserts with instructions for use.